In recent, there has been developed a real-time PCR technology that can real-time monitor a reaction product during PCR process. With this technology, it is not necessary to perform an electrophoresis on gel and it is possible to confirm the amplified product during the reaction cycle as well as to obtain a quantitative result. In order to perform this real-time PCR, an apparatus incorporated with a thermal cycler for the PCR reaction and a fluorometer for the real-time detection of reactant is used.
In general, a fluorescent detection using a fluorescent reagent is used to monitor the real-time PCR, typical methods includes the followings:
1) Intercalating method: an Intercalator (for example, SYBR Green I, EtBr, etc.), i.e. a reagent that shows fluorescence by binding with double strand DNA is added to a reaction system and fluorescence generated with amplification is detected. That is to say, fluorescence is generated when an Intercalator is bound with double strand DNA synthesized through the PCR and it is possible to measure quantity as well as melting temperature of the amplified DNA by detecting the fluorescent intensity.
2) TaqMan™ probe method: oligonucleotide in which 5′ terminal is modified to fluorescent material (e.g. FAM, etc.) and 3′ terminal is modified to quencher material (e.g. TAMRA, etc.) is added. Under an annealing condition, TaqMan™ probe is specifically hybridized with a template DNA, but fluorescence is blocked by the quencher. During an amplification reaction, fluorescence generated as the template is digested by 5′-3′ exonuclease activity of the Taq DNA polymerase and then the blocking by capture is released.
3) Molecular Beacon method: oligonucleotide (Molecular Beacon probe) that forms a secondary hairpin structure in which both ends thereof are modified to fluorescent material (e.g. FAM, TAMRA, etc.) and quencher material (e.g. DABCYL, etc.) is added to the reaction. The Molecular Beacon probe is specifically hybridized with a complementary region to a template under an annealing condition. At this time, fluorescence generated as a distance between the fluorescent dye and the quencher becomes more distant and the blocking by the quencher is thus released. Meanwhile, unhybridized Molecular Beacon probe does not generate the fluorescence since it has a secondary structure and is thus blocked by the quencher.
A conventional apparatus for real-time PCR (U.S. Pat. No. 6,818,437) includes, as shown in FIG. 1, a thermoelectric element 1c, a block 1 for transferring heat to reaction tubes 2a containing a sample, a light source 11 for irradiating a beam into the sample contained in the reaction tube and a sensor part 78 for receiving fluorescence generated from the sample. The principle of the aforementioned apparatus is as follows: in order to react nucleic sample solution within the tube, a cooling and heating cycle is repeatedly performed using the thermoelectric element 1c; and upon completion of each cycle, an intensity of the fluorescence generated from the sample is measured by the operation of the light source 11 and the sensor part 78, thereby checking the progress of the reaction in real-time. The light source 11 is a white light source, and a band pass filter 7 is used in order to generate an excitation light having a frequency corresponding to that of the used fluorescent probe. A dichroic beam splitter 6 is a device for separating the excitation light and the fluorescence. In FIG. 1, it reflects light having a frequency lower than a specific frequency and passes light having a frequency higher than the specific frequency. Another band pass filter 8 is for selectively passing only the fluorescence emitted from the sample to the sensor part 78. In addition, a Fresnel lens 3 is used for parallelizing the excitation light.
In recent, there has been introduced a real-time PCR experiment in which fluorescent probes of various colors are used at the same time. However, in the conventional technology, different dichroic beam splitters are required according to the fluorescent probe if the frequencies of the used fluorescent probes are different. Therefore, in the conventional technology, the band pass filters 7 and 8 and the dichroic beam splitters 6 are incorporated into a single module, and data is obtained while changing the module according to the fluorescent probe. For example, if five kinds of band pass filters are used, five kinds of dichroic beam splitters are required to match the band pass filters.
Also, in the case of the dichroic beam splitter used in the conventional technology, since the excitation light is generally brighter 105 times then the fluorescence generated from the sample, it is impossible to completely separate the excitation light and the fluorescence. Further, a reflection light of the excitation light by an optical component on the light path is incident to the fluorescence detecting part and interferes with the fluorescence generated from the sample.
The factor that causes the reflection of the excitation light includes:
1) the Fresnel lens 3 used to parallelize the excitation light, 2) a lid 2b or a transparent tape used to prevent vaporization of the sample in the reaction tube 2a and 3) the reaction tube itself.